Elucidating microbial iron corrosion mechanisms with a hydrogenase‐deficient strain of Desulfovibrio vulgaris

Abstract Sulfate‐reducing microorganisms extensively contribute to the corrosion of ferrous metal infrastructure. There is substantial debate over their corrosion mechanisms. We investigated Fe0 corrosion with Desulfovibrio vulgaris, the sulfate reducer most often employed in corrosion studies. Cultures were grown with both lactate and Fe0 as potential electron donors to replicate the common environmental condition in which organic substrates help fuel the growth of corrosive microbes. Fe0 was corroded in cultures of a D. vulgaris hydrogenase‐deficient mutant with the 1:1 correspondence between Fe0 loss and H2 accumulation expected for Fe0 oxidation coupled to H+ reduction to H2. This result and the extent of sulfate reduction indicated that D. vulgaris was not capable of direct Fe0‐to‐microbe electron transfer even though it was provided with a supplementary energy source in the presence of abundant ferrous sulfide. Corrosion in the hydrogenase‐deficient mutant cultures was greater than in sterile controls, demonstrating that H2 removal was not necessary for the enhanced corrosion observed in the presence of microbes. The parental H2‐consuming strain corroded more Fe0 than the mutant strain, which could be attributed to H2 oxidation coupled to sulfate reduction, producing sulfide that further stimulated Fe0 oxidation. The results suggest that H2 consumption is not necessary for microbially enhanced corrosion, but H2 oxidation can indirectly promote corrosion by increasing sulfide generation from sulfate reduction. The finding that D. vulgaris was incapable of direct electron uptake from Fe0 reaffirms that direct metal‐to‐microbe electron transfer has yet to be rigorously described in sulfate‐reducing microbes.


INTRODUCTION
The worldwide economic cost of microbial corrosion is likely to exceed a trillion dollars a year, and current strategies for mitigating corrosion are relatively ineffective [1][2][3] .A better understanding of the factors controlling microbial corrosion rates might provide insights for the development of new strategies to prevent corrosion.The microbial impact on the corrosion of ferrous metals is typically most substantial under anaerobic conditions.In anaerobic corrosion, Fe 0 is oxidized to Fe 2+ : A diversity of microbes can establish electrical contacts with Fe 0 , directly accepting electrons derived from Fe 0 oxidation to 1 Electrobiomaterials Institute, Key Laboratory for Anisotropy and Texture of Materials (Ministry of Education), Northeastern University, Shenyang, China. 2 Shenyang National Laboratory for Materials Science, Northeastern University, Shenyang, China. 3Department of Microbiology, University of Massachusetts, Amherst, MA, USA.support anaerobic respiration.Microbes capable of this "electrobiocorrosion" 2 include Geobacter 4,5 , Shewanella 6,7 , Methanosarcina 8,9 , Methanothrix 9 , and Sporomusa 9 species.Known electron acceptors for electrobiocorrosion are nitrate, Fe(III), and carbon dioxide.Although the sulfate-reducing microbes Desulfovibrio ferrophilus and Desulfopila corrodens were proposed to be electrobiocorrosive 10 , subsequent studies found that they were not 11 .
However, sulfate reducers are often the most abundant and metabolically active microbes associated with intense anaerobic corrosion [12][13][14][15][16][17] .The most abundant sulfate reducers are often Desulfovibrio species [18][19][20] .The mechanisms by which Desulfovibrio and related sulfate reducers promote corrosion have been a matter of debate since some of the earliest studies of microbial corrosion [19][20][21][22][23][24][25][26][27][28] .Those early studies often focused on Desulfovibrio H 2 uptake because, even in the absence of microbes, an important route for anaerobic Fe 0 oxidation is proton reduction: Although it is clear that Fe 0 oxidation can serve as a H 2 source for H 2 -oxidizing anaerobes, including Desulfovibrio species (see Ueki and colleagues 19,29 for extensive lists), there has been substantial debate whether microbial H 2 consumption can accelerate Fe 0 oxidation coupled to H 2 production [20][21][22][23][24]26,27,30,31 . Studies conucted under abiotic conditions have indicated that removal of H 2 should have no effect on the rate of Fe 0 oxidation with H + reduction and that this reaction is not inhibited as H 2 concentrations increase 26 .Arguments for an important role of microbial H 2 uptake have often merely demonstrated or inferred that H 2 produced from Fe 0 oxidation was microbially consumed but did not rigorously demonstrate that H 2 uptake accelerated Fe 0 oxidation 20,[22][23][24] .
An example of the difficulty in interpreting the studies on this topic are two studies with the same first author submitted within 10 days of each other 24,32 .In the first study submitted, cell suspensions of H 2 -consuming Desulfovibrio desulfuricans provided with fumarate as the electron acceptor did not accelerate the weight loss of mild steel over that observed in sterile controls 24 .However, the second study reported the results of electrochemical studies conducted with cell suspensions of Desulfovibrio vulgaris with benzyl viologen as the electron acceptor that suggested that microbes with hydrogenases could accelerate Fe 0 oxidation 32 .
A complication in evaluating the role of H 2 uptake in accelerating Fe 0 oxidation is that the sulfide produced during sulfate reduction can enhance H 2 production from Fe 0 .Iron sulfides formed from the Fe 2+ from Fe 0 oxidation and sulfide, can promote reaction #1 when deposited on Fe 0 surfaces 27,28

+ + ( )
Another potential complicating factor has been the suggestion that iron sulfides might enable direct Fe 0 -to-microbe electron transfer, that is, electrobiocorrosion 33 .This hypothesis was derived from electrochemical studies.Cathodic currents were generated with electrodes poised at (−0.4 V) in the presence of D. vulgaris with iron sulfide nanoparticles on the cell surface but not in the absence of the iron sulfide-coated cells 33 .This result was interpreted as direct electrode-to-microbe electron transfer.However, iron sulfides can readily lower electrode overpotentials for the reduction of H + to H 2 24,34   , and may stimulate H 2 uptake by sulfate reducers 34 .These potential alternative explanations for the higher cathodic currents in the presence of the iron sulfide-coated cells were not rigorously ruled out.These considerations, and the fact that the role of microbially produced ferrous sulfides on Fe 0 oxidation was speculated, not experimentally demonstrated, indicates that more rigorous evaluation of the routes for Fe 0 corrosion in the presence of iron sulfides is required.
An effective approach for evaluating the role of H 2 uptake in microbial corrosion is to construct strains in which the genes for hydrogenases have been deleted [4][5][6]35 . Forexample, studies with strains of Geobacter sulfurreducens and Shewanella oneidensis in which the genes for the H 2 uptake hydrogenases were deleted demonstrated that these microbes accepted electrons from Fe 0 even as H 2 accumulated 4,6 .However, these microbes were also capable of electrobiocorrosion, providing an alternative route for electron uptake from Fe 0 4,6 .In contrast, a hydrogenase-deficient mutant of D. vulgaris unable to utilize H 2 did not reduce sulfate with Fe 0 as the electron donor, whereas the parental strain did 35 .This result demonstrated that D. vulgaris relied on H 2 as an intermediary electron carrier between Fe 0 and cells and was incapable of electrobiocorrosion when only Fe 0 was available as the electron donor and energy source.However, some methanogens are only effective in electrobiocorrosion when they are metabolizing an additional energy source as they oxidize Fe 0 8,9 .The possibility that D. vulgaris might also directly accept electrons from Fe 0 in the presence of an additional energy source was not previously evaluated 35 .This is important because microbes also have access to organic substrates in most environments in which metals are being corroded 2 .
Here, we report on Fe 0 corrosion studies with the same hydrogenase-deficient strain of D. vulgaris employed in previous studies 35 , but this time grown with lactate as an additional electron donor.The results demonstrate that microbial consumption of H 2 was not essential for extensive corrosion during sulfate reduction and that the hydrogenasedeficient strain of D. vulgaris was incapable of electrobiocorrosion, even with extensive ferrous sulfide production and the additional lactate energy source.Multiple lines of evidence were consistent with the concept that microbial sulfide production stimulates Fe 0 oxidation with the production of H 2 .

RESULTS AND DISCUSSION
Biofilm growth D. vulgaris was grown in the presence of lactate and Fe 0 as potential energy sources because organic substrates are available to microbes in most anaerobic corrosive environments 2 and because the hydrogenase-deficient mutant does not grow with Fe 0 as the sole electron donor 35 .The parental strain (JW710) and the hydrogenase-deficient mutant strain (JW5095) grew at similar rates in the lactate-sulfate medium employed in this study 35 .When Fe 0 was included in the lactate-sulfate medium, both strains produced substantial biofilms on the Fe 0 surfaces (Figure 1).

Lack of direct electron transfer and hydrogenase impact on corrosion
As expected from previous studies 35 , H 2 accumulated in the hydrogenase-deficient mutant cultures, but not with the parental strain (Figure 2A).The H 2 produced (Figure 2A) and the Fe 0 loss (Figure 2B) in the mutant cultures were threefold higher than previously reported in sulfide-free sterile incubations under the same conditions 36 .This result is consistent with the concept that as microbes reduce sulfate to sulfide, the sulfide promotes Fe 0 oxidation with the reduction of H + to H 2 .Although some microbes release hydrogenases that can promote Fe 0 oxidation with the generation of H 2 37,38 , this possibility can be ruled out in our studies because the hydrogenase genes had been deleted from the mutant and even the parental strain does not release extracellular hydrogenases 35 .Multiple lines of evidence indicated a lack of direct Fe 0 -tomicrobe electron transfer 2 .When Fe 0 is oxidized via direct electron transfer to microbes, no H 2 is produced.However, within the error of the measurements, the accumulation of H 2 in the hydrogenase-deficient mutant cultures (0.061 ± 0.0028 mmol (mean ± standard deviation, n = 3; Figure 2A) was comparable to the loss of Fe 0 (0.070 ± 0.012 mmol; Figure 2B), as expected from the 1:1 correspondence between Fe 0 oxidation and H 2 production when Fe 0 is oxidized with the reduction of H + (reaction #2) or hydrogen sulfide reacts with Fe 0 (reaction #3).Furthermore, if the hydrogenase-deficient strain had derived electrons from direct electron transfer, then those electrons would have contributed to sulfate reduction.Yet, when it is considered that ca.10% of the organic substrate supporting sulfate reduction is diverted to biosynthesis 39,40 , the amount of sulfate reduction (0.067 ± 0.011 mmol of sulfate reduced; Figure 2C) compared well with the 0.0675 mmol of sulfate expected to be reduced with the 5 mM lactate that was provided as an electron donor (5 mmol lactate/l × 0.9 [fraction consumed in respiration] × 0.03 l of media × 1 mmol sulfate reduced/2 mmol lactate oxidized to acetate = 0.0675 mmol sulfate reduced).Thus, the Fe 0 loss/H 2 accumulation stoichiometry and the extent of sulfate reduction demonstrated a lack of D. vulgaris direct electron uptake even though lactate was available as an additional energy source and abundant ferrous sulfide was produced.

Dual electron sources for the parental strain
The parental strain reduced more sulfate than the mutant strain (Figure 2C) and substantially more than the sulfate reduction possible with the 0.075 mmol of lactate available.Thus, unlike the mutant strain, the parental strain benefited from electrons derived from Fe 0 oxidation.The evidence suggests that these electrons were transferred to the parental strain via a H 2 intermediate.The low H 2 levels in the parental strain cultures (Figure 2A) demonstrated that the parental strain consumed the H 2 that accumulated in the mutant cultures.Just the parental strain consumption of that excess H 2 alone could account for 65% of the additional sulfate that the parental strain reduced because: 1.The additional sulfate reduction by the parental strain was 0.023 mmol (0.090 mmol sulfate reduced by the parental strain -0.067 mmol sulfate reduced by the mutant strain = 0.023 mmol); 2. The parental strain consumed the 0.061 mmol of H 2 that accumulated in the mutant cultures; Furthermore, the greater sulfide production resulting from the parental strain oxidizing H 2 could be expected to further stimulate Fe 0 oxidation with the production of even more H 2 .This was apparent from the greater weight loss of Fe 0 (Figure 2B) and deeper pitting in the parental strain cultures (Figure 2D-H).The 0.008 mmol of sulfate reduction that could not be accounted for from the consumption of the H 2 accumulation in the mutant cultures is the equivalent of 0.032 mmol of Fe 0 oxidized (4 mmol Fe 0 oxidized per mmol of sulfate reduced).Within the error of the measurements, 0.032 mmol Fe 0 oxidized is comparable to the difference in the weight loss of the parental (0.107 ± 0.018) and mutant (0.070 ± 0.012) strains.As detailed above, extracellular hydrogenases and direct electron uptake could be ruled out as potential mechanisms for promoting sulfate reduction.Thus, the positive feedback loop of sulfate reduction generating sulfide, resulting in more H 2 production to support more sulfate reduction, is the most likely explanation for the greater corrosion in the parental strain cultures.

Similar corrosion mechanism in both strains
The open circuit potential (OCP) during Fe 0 corrosion was similar for both strains (Figure 3A), as would be expected if Fe 0 oxidation was proceeding with H 2 production with both strains.This result also suggested that the accumulation of H 2 in the hydrogenase mutant did not substantially inhibit Fe 0 oxidation reactions.Corrosion resistance was only slightly higher with the hydrogenase-deficient strain (Figure 3B), whereas corrosion current, calculated from the potentiodynamic polarization (Figure 3C), was slightly lower (Figure 3D).These results reflected the somewhat greater corrosion with the parental strain (Figure 2B), which as discussed above, can be most likely attributed to greater sulfide deposition with the parental strain.

Implications
This study illustrates the potential for rigorously evaluating often contentious mechanistic claims in the corrosion literature with mutant strains.The availability of a hydrogenasedeficient mutant enabled studies that clearly demonstrate that D. vulgaris is incapable of discernable direct electron uptake from Fe 0 , even when supplied with lactate as an additional energy source and in the presence of abundant ferrous sulfide.The lack of D. vulgaris direct electron uptake from Fe 0 emphasizes that the claim for D. vulgaris direct electron uptake from electrodes in the presence of ferrous sulfides 33 should also be reexamined with the hydrogenasedeficient mutant.
The substantial corrosion in cultures of the hydrogenasedeficient mutant provides evidence from a metabolically active system that H 2 consumption does not promote Fe 0 oxidation coupled to H + reduction, a conclusion consistent with previous findings in abiotic studies 26 .H 2 partial pressures of more than 2044 atm (207 Mpa), a value much higher than conceivable in any natural environment, would have been required to make Fe 0 oxidation coupled to H 2 production (reaction #2) thermodynamically unfavorable in our incubations (see Material for calculations in Supporting Information).
Thus, H 2 -utilizing sulfate reducers benefit from Fe 0 corrosion but do not directly promote corrosion with H 2 removal.The primary importance of microbial H 2 consumption is that it provides an electron donor for additional sulfate reduction to sulfide.This was evidenced by the higher corrosion in the presence of the parental strain versus the hydrogenasedeficient mutant.
The mechanisms for microbial Fe 0 corrosion have only been rigorously examined with functional genetic studies in a few model microbes [4][5][6][7]35 . Furhermore, other physiological factors such as mechanisms for biofilm formation and how outer-surface cellular components influence Fe 0 -microbe interactions and corrosion have yet to be investigated with in-depth molecular approaches.It is also unknown how well the microbes available in culture represent those that make the largest contributions to Fe 0 corrosion outside the laboratory 29 .A concerted attempt to recover highly corrosive microbes in culture and elucidate how they corrode with definitive genetic and biochemical studies is required if microbial corrosion is to be understood at the level necessary to innovate new corrosion mitigation strategies.

Strains and growth conditions
Two D. vulgaris strains were supplied by Valentine V. Trotter and Adam M. Deutschbauer of the Lawrence Berkeley Laboratory.Strain JW710, also referred to here as the parental strain, is the strain employed for a markerless genetic exchange system in D. vulgaris 41 .Markerless deletion of all of the known hydrogenase genes in strain JW710 yielded strain JW5095 42 .Previous studies 35 demonstrated that the two strains grew equally well with lactate as the electron donor and sulfate as the electron acceptor.The parental strain JW710 also grew with H 2 as the sole electron donor, whereas JW5095 did not utilize H 2

35
. Cultures were routinely maintained in the previously described defined NB medium 35 under N 2 /CO 2 (80:20) with lactate (60 mM) as the electron donor and sulfate (30 mM) as the electron acceptor.Standard anaerobic culture procedures, including maintaining cultures under oxygen-free gases, sealing culture vessels with thick butyl rubber stoppers, and transferring and sampling cultures with needles and syringes, were employed throughout.
For corrosion studies, the cultures were provided with sulfate (5 mM) as the electron acceptor and lactate (5 mM) and three Fe 0 coupons (8 mm × 8 mm × 5 mm; wt% composition: Fe 99.95, C 0.01, S 0.008, P 0.01, Si 0.001, Mn 0.001, Ni 0.001, Cr 0.01, and Al 0.006) as potential electron donors in 30 ml of medium in 65 ml serum bottles.Before using, the Fe 0 coupons were polished with a sequence of 240, 600, and 1000 grit sandpaper and then washed with ethanol and MilliQ water.The polished coupons were sterilized by soaking them in 75% ethanol and drying them in a UV chamber and then aseptically and anaerobically added to the medium.Incubations were at 30°C.

Analytical methods
H 2 concentrations in the culture headspaces were determined with gas chromatograph with a thermal conductivity detector (Trace 1310; Thermo Scientific).Gases were separated with J&W CP-Molsieve 5 Å column (60 m × 0.32 mm, Agilent Technologies, Inc.) with N 2 carrier gas.For sulfate analysis, liquid samples were diluted in water 20-fold, filtered (0.22 μm pore diameter polyvinylidene difluoride membrane), and analyzed with an ion chromatograph (Dionex AS-DV; Thermo Scientific) with an AS11-HC (4 × 250 mm) analytical column and AG11-HC (4 × 50 mm) guard with an eluent of potassium hydroxide at 1.0 ml/min.

Biofilm visualization
For visualization of biofilms with scanning electron microscopy, the Fe 0 coupons were rinsed with pH 7.4 phosphatebuffered solution (PBS) and fixed with glutaraldehyde (2.5% v/v) for 2 h.The samples were then successively dehydrated with 50%, 60%, 70%, 80%, and 90% ethanol solutions for 10 min and then 100% ethanol solution for 5 min before gold-coating the samples and examining them with an EVO 10 scanning electron microscope (Zeiss).
For biofilm visualization with confocal scanning laser microscopy, the Fe 0 coupons were washed with PBS and then stained with the Live/Dead BacLight™ bacterial staining kit (Molecular Probes L-7012; Thermo Fisher Scientific) for 10 min in the dark.The samples were examined with the fluorescent channels of an LSM900 confocal laser scanning microscope (Zeiss).

Analysis of corrosion weight loss and pitting
To determine Fe 0 weight loss, the coupons were treated with Clarke's solution (1000 ml hydrochloric acid [specific gravity 1.19], 20 g antinomy trioxide [Sb 2 O 3 ], and 50 g stannous chloride [SnCl 2 ]) following the ASTM G1-03 protocol 43 to remove corrosion products.The treated surfaces were scanned with the LSM900 microscope in bright-field mode, and the data were analyzed with ConfoMap Premium Software (Zeiss) to determine corrosion pitting depth, surface morphologies, and pitting profiles.

Electrochemical characterization
Electrochemical parameters of corrosion were determined with a classical three-electrode system with a platinum plate (10 mm × 10 mm × 1 mm) as the counter electrode, a saturated calomel electrode as the reference electrode, and a Fe 0 electrode with a 1 × 1 cm exposed surface as the working electrode.The OCP and linear polarization resistance (LPR) were monitored daily.Potentiodynamic polarization (PDP) was determined after 7 days of incubation.The electrochemical tests were conducted with electrochemical workstations (Reference 600; Gamry Instruments).The LPR tests were scanned with a rate of 0.167 mV/s in the range of −10 to 10 mV versus OCP.PDP were conducted at a scan rate of 0.333 mV/s from −0.3 to 0.3 V versus OCP.

Figure 1 .
Figure 1.Biofilm growth on Fe 0 surfaces in the lactate-sulfate medium.(A, B) Scanning electron microscopy images of the biofilms of the parental strain JW710 (A) and hydrogenase-deficient strain JW5095 (B) after fixation and gold coating.(C, D) Confocal laser scanning microscope visualization of parental strain JW710 (C) and hydrogenase-deficient strain JW5095 (D) after staining with Live/Dead BacLight™ bacterial staining kit.Large central images show x-y plane of the Desulfovibrio vulgaris biofilm, small top and right-side images show x-z and y-z planes of the biofilm, respectively.

Figure 2 .
Figure 2. Metabolic and corrosion parameters of the parental strain (JW710) and hydrogenase-deficient strain (JW5095) during growth in lactate-sulfate medium in the presence of Fe 0 after 7 days of incubation.(A) H 2 accumulation.(B) Loss of Fe 0 .(C) Sulfate consumption.(D) Pit parameters from the 10 deepest corrosion pits of the two strains.(E-H) Surface morphology (E, F) and profiles (G, H) of the deepest pits for each strain.Values in A-C are the mean and standard deviation of triplicate incubations.

Figure 3 .
Figure 3. Electrochemical analysis of corrosion with the parental strain (JW710) and hydrogenase-deficient strain (JW5095).(A) Open circuit potential (OCP) during 7-day incubations.(B) Polarization resistance (R p ) during 7-day incubations.(C) Potentiodynamic polarization curves of potential (E in volts [V]) vs. saturated calomel electrode (SCE) vs. log 10 of current (i) on Day 7. (D) Corrosion current density (i corr ) calculated from potentiodynamic polarization curves on Day 7. Error bars represent the standard deviation of the mean of triplicate incubations.